Stepwise photoinhibition of photosystem II. Studies with Synechocystis species PCC 6803 mutants with a modified D-E loop of the reaction center polypeptide D1

Abstract

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant DeltaR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants DeltaG240-V249 and DeltaR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the DeltaG240-V249 and DeltaR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.

Figure 1

Photoinactivation of PSII oxygen evolution during illumination of AR (□) and mutant cells (PD, ▵; CD, ○; and PCD, ) of Synechocystis sp. PCC 6803. Results are the means of three to five independent experiments from different cell cultures, and are expressed as a percentage of oxygen-evolution activity measured before the high-light treatment. Bars denote se. A, Cells were illuminated at a PPFD of 1500 μmol m⁻² s⁻¹ and oxygen evolution was measured in vivo using DCBQ as the electron acceptor. B, Cells were illuminated at a PPFD of 1000 μmol photons m⁻² s⁻¹ and oxygen evolution was assayed in vivo with DCBQ as the electron acceptor. C, Cells were illuminated at a PPFD of 1000 μmol photons m⁻² s⁻¹ and oxygen evolution was assayed in vivo with DMBQ as the electron acceptor. D, The PD and AR cells were illuminated at a PPFD of 500 μmol photons m⁻² s⁻¹ in the presence of lincomycin (400 μg/mL) and oxygen evolution was monitored with DCBQ (□, ○) or with bicarbonate (▪, •) as the electron acceptor.

Figure 2

Recovery of PSII from photoinhibition. Protein-synthesis-independent (A and B) and protein-synthesis-dependent (C) restoration of PSII oxygen evolution in Synechocystis PCC 6803 strains after photoinhibitory illumination. Results are means of three independent experiments from different cell cultures and bars denote se. A, AR (□) and PD (▵) cells were illuminated at 1000 μmol photons m⁻² s⁻¹ for 60 min (PI) in the absence of lincomycin. The subsequent recovery of oxygen evolution was followed in the presence of lincomycin at 40 μmol photons m⁻² s⁻¹ and was monitored with DCBQ as the electron acceptor. B, The AR (□) and PD (▵) cells were illuminated at 1000 μmol photons m⁻² s⁻¹ for 60 min (PI) in the absence of lincomycin. The subsequent recovery of oxygen evolution at 40 μmol photons m⁻² s⁻¹ in the presence of lincomycin was monitored using bicarbonate as the electron acceptor. C, AR (□), PD (▵), CD (○), and PCD () strains were illuminated at 1500 μmol photons m⁻² s⁻¹ for 60 min (PI), and the restoration of DCBQ-dependent oxygen evolution was followed at 40 μmol photons m⁻² s⁻¹. All treatments were performed in the absence of lincomycin.

Figure 3

Chase of radioactivity in membrane polypeptides of Synechocystis sp. PCC 6803 mutants. The cells were first pulse labeled for 75 min in the presence of l-[³⁵S]Met, and then radioactivity was chased for 0, 15, 30, 45, and 60 min at 1500 μmol photons m⁻² s⁻¹. Arrowheads indicate the location of the mutated D1 polypeptides. Correct identification of the radiolabeled D1 polypeptide was confirmed by western blotting.

Figure 4

Accumulation of the psbA-2 mRNA in the AR, PCD, and PD cells in the course of high-light illumination (1500 μmol photons m⁻² s⁻¹) in the absence or presence of lincomycin, as indicated. The amount of RNA is given in arbitrary units comparable between the strains. White bars, 0 min of illumination; stippled bars, 30 min of illumination; and black bars, 60 min of illumination.

Figure 5

Representative northern blots showing the psbA-2 mRNA levels in the AR strain during photoinhibitory illumination of 1500 μmol photons m⁻² s⁻¹ in the absence (A) and presence (B) of the protein-synthesis inhibitor lincomycin. The bottom panels present the same membranes probed with rrn genes of A. nidulans. Five micrograms of total RNA was loaded in each well.